We demonstrate that in Saccharomyces cerevisiae, the tandem array of ribosomal RNA genes [RDNl) is a target for integration of the Tyl retrotransposon that resuhs in silencing of Tyl transcription and transposition. Tyl elements transpose into random rDNA repeat units and are mitotically stable. In addition, we have found that mutation of several putative modifiers of RDNl chromatin structure abolishes silencing of Tyl elements in the rDNA array. Disruption of SIR2, which elevates recombination in RDNl, or TOPI, which increases psoralen accessibility in rDNA, or HTAl-HTBl, which reduces histone H2A-H2B levels and causes localized chromatin perturbations, abolishes transcriptional silencing of Tyl elements in RDNl. Furthermore, deletion of the gene for the ubiquitin conjugating enzyme Ubc2p, which ubiquitinates histones in vitro, derepresses not only Tyl transcription but also mitotic recombination in RDNl. On the basis of these results, we propose that a specialized chromatin structure exists in RDNl that silences transcription of the Tyl retrotransposon.
The molecular identity of a gene which encodes the pore-forming subunit (alpha1G) of a member of the family of low-voltage-activated, T-type, voltage-dependent calcium channels has been described recently. Although northern mRNA analyses have shown alpha1G to be expressed predominantly in the brain, the detailed cellular distribution of this protein in the central nervous system (CNS) has not yet been reported. The current study describes the preparation of a subunit specific alpha1G riboprobe and antiserum which have been used in parallel in situ mRNA hybridization and immunohistochemical studies to localize alpha1G in the mature rat brain. Both alpha1G mRNA and protein were widely distributed throughout the brain, but variations were observed in the relative level of expression in discrete nuclei. Immunoreactivity for alpha1G was typically localized in both the soma and dendrites of many neurons. Whilst alpha1G protein and mRNA expression were often observed in cells known to exhibit T-type current activity, some was also noted in regions, e.g. cerebellar granule cells, in which T-type activity has not been described. These observations may reflect differences between the subcellular distribution of channels that can be identified by immunohistochemical methods compared with electrophysiological techniques.
Ty1 retrotransposons in Saccharomyces cerevisiae are maintained in a state of transpositional dormancy. We isolated a mutation, rtt100-1, that increases the transposition of genomic Ty1 elements 18-to 56-fold but has little effect on the transposition of related Ty2 elements. rtt100-1 was shown to be a null allele of the FUS3 gene, which encodes a haploid-specific mitogen-activated protein kinase. In fus3 mutants, the levels of Ty1 RNA, protein synthesis, and proteolytic processing were not altered relative to those in FUS3 strains but steady-state levels of TyA, integrase, and reverse transcriptase proteins and Ty1 cDNA were all increased. These findings suggest that Fus3 suppresses Ty1 transposition by destabilizing viruslike particle-associated proteins. The Fus3 kinase is activated through the mating-pheromone response pathway by phosphorylation at basal levels in naive cells and at enhanced levels in pheromone-treated cells. We demonstrate that suppression of Ty1 transposition in naive cells requires basal levels of Fus3 activation. Substitution of conserved amino acids required for activation of Fus3 derepressed Ty1 transposition. Moreover, epistasis analyses revealed that components of the pheromone response pathway that act upstream of Fus3, including Ste4, Ste5, Ste7, and Ste11, are required for the posttranslational suppression of Ty1 transposition by Fus3. The regulation of Ty1 transposition by Fus3 provides a haploid-specific mechanism through which environmental signals can modulate the levels of retrotransposition.Retroviruses and endogenous retrovirus-like elements are ubiquitous in the eucaryotic kingdom and have been involved in the formation of a significant portion of the typical eucaryotic genome (45). Hence, eucaryotes have evolved many types of regulatory mechanisms to control the replication and mobility of retroelements. However, only a few host genes that control retroviral replication in vertebrates have been identified. A recent example is the mouse Fv1 gene, whose product is derived from the gag domain of an endogenous retroelement and is postulated to inhibit murine leukemia virus replication by interacting with the viral capsid protein (4).Aside from the infectivity of the retroviral particle, the steps of retrotransposition are analogous to retroviral replication. A well-characterized model system to study host regulation of retrovirus-like elements is the Ty1 retrotransposon in the yeast Saccharomyces cerevisiae (8,50). Ty1 elements have two long terminal repeats (LTRs) surrounding a central region consisting of two overlapping open reading frames: TyA, which encodes a structural capsid protein, and TyB, which encodes protease (PR), integrase (IN), and reverse transcriptase (RT) activities. Replication of Ty1 occurs in the following sequence of events: a chromosomal Ty1 element is transcribed from LTR to LTR by RNA polymerase II into a terminally redundant RNA that is polyadenylated and transported to the cytoplasm. TyA and TyA-TyB fusion protein are synthesized from the full-leng...
Ty1 retrotransposons in the yeast Saccharomyces cerevisiae are maintained in a genetically competent but transpositionally dormant state. When located in the ribosomal DNA (rDNA) locus, Ty1 elements are transcriptionally silenced by the specialized heterochromatin that inhibits rDNA repeat recombination. In addition, transposition of all Ty1 elements is repressed at multiple posttranscriptional levels. Here, we demonstrate that Sgs1, a RecQ helicase required for genome stability, inhibits the mobility of Ty1 elements by a posttranslational mechanism. Using an assay for the mobility of Ty1 cDNA via integration or homologous recombination, we found that the mobility of both euchromatic and rDNA-Ty1 elements was increased 32-to 79-fold in sgs1⌬ mutants. Increased Ty1 mobility was not due to derepression of silent rDNA-Ty1 elements, since deletion of SGS1 reduced the mitotic stability of rDNA-Ty1 elements but did not stimulate their transcription. Furthermore, deletion of SGS1 did not significantly increase the levels of total Ty1 RNA, protein, or cDNA and did not alter the level or specificity of Ty1 integration. Instead, Ty1 cDNA molecules recombined at a high frequency in sgs1⌬ mutants, resulting in transposition of heterogeneous Ty1 multimers. Formation of Ty1 multimers required the homologous recombination protein Rad52 but did not involve recombination between Ty1 cDNA and genomic Ty1 elements. Therefore, Ty1 multimers that transpose at a high frequency in sgs1⌬ mutants are formed by intermolecular recombination between extrachromosomal Ty1 cDNA molecules before or during integration. Our data provide the first evidence that the host cell promotes retrotransposition of monomeric Ty1 elements by repressing cDNA recombination.DNA helicases catalyze the unwinding of duplex DNA into individual DNA strands (42). A plethora of DNA helicases within cells is involved in DNA replication, repair, recombination, and transcription. Members of the RecQ family of DNA helicases are involved in the maintenance of genome stability in all organisms characterized, from bacteria to humans (7). Mutations in the SGS1 gene, which encodes the only RecQ homologue in Saccharomyces cerevisiae, result in elevated levels of mitotic homologous and illegitimate recombination, increased rates of chromosomal nondisjunction, and accelerated aging (21,56,61,62,65). Similarly, mutations in human genes encoding the RecQ homologues RecQL4 (35, 49), WRN (67), and BLM (18) give rise to rare hereditary disorders that are characterized by genome instability and a pronounced predisposition to cancer. Notably, expression of either WRN or BLM in yeast complements the hyperrecombination phenotypes of an sgs1 mutant (65). These findings suggest that the mechanisms by which Sgs1 preserves genetic stability in yeast will serve as a paradigm for the role of RecQ homologues in human disease.The SGS1 gene was originally isolated in a screen for genetic interaction with DNA topoisomerase III (21). Both topoisomerase III and Sgs1 repress recombination of DNA repe...
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